Cyclic tetrapeptide stereoisomers

ABSTRACT

Cyclic tetrapeptide stereochemical isomers of CJ-15,208, pharmaceutical compositions from such cyclic tetrapeptides, and methods of using such pharmaceutical compositions. The cyclic tetrapeptide compounds and pharmaceutical compositions disclosed herein are potent analgesics active in several pain models with generally minimal tolerance and reduced likelihood to induce addiction relative to other known opiates.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a U.S. Nationalization of PCT Application NumberPCT/US2015/040184, filed on Jul. 13, 2015, which claims the benefit ofand priority to U.S. Prov. App. Ser. No. 62/023,392 filed 11 Jul. 2014,the entireties of which are incorporated herein by reference in theirentireties.

GOVERNMENT RIGHTS

This invention was made with government support under Grant Numbers R01DA018832 and R01 DA023924 awarded by the National Institute of Health.The government has certain rights in this invention.

BACKGROUND

Opioid receptors are a group of inhibitory G protein-coupled receptorswith to opioids as ligands. Opioid receptors are distributed widely inthe brain, and are found in the spinal cord and digestive tract. Thetypes of opioid receptors are mu (μ) MOR, kappa (κ) KOR, and delta (δ)DOR. The receptors were generally named using the first letter of thefirst ligand that was found to bind to them. For instance, morphine wasthe first chemical shown to bind to mu receptors. The first letter ofthe drug morphine is m, rendered as the corresponding Greek letter μ. Insimilar manner, a drug known as ketocyclazocine was first shown tointeract with kappa receptors, while the delta receptor was named afterthe mouse vas deferens tissue in which the receptor was firstcharacterized. The mu receptor is the primary target of currently usednarcotic analgesics.

The opioid receptor types are ˜70% identical with substantialdifferences located at N and C termini as well as other regions of thereceptors. It is thought that the G protein binds to the thirdintracellular loop of the opioid receptors. Both in mice and humans, thegenes for the various receptor subtypes are located on differentchromosomes.

Some characteristics of the MOR, KOR, and DOR are presented below.

The mu (μ) opioid receptor (MOR) is found in the brain (e.g., in thecortex (laminae III and IV), thalamus, striosomes, periaqueductal gray,and rostral ventromedial medulla), in the spinal cord (e.g., in thesubstantia gelatinosa), in the peripheral sensory neurons, and in theintestinal tract. The mu receptor has roles in analgesia, physicaldependence, respiratory depression, miosis, euphoria, reducedgastrointestinal motility, physical dependence, and possiblevasodilation.

The kappa (κ) opioid receptor (KOR) is found in the brain (e.g., in thehypothalamus, periaqueductal gray, and claustrum), in the spinal cord(e.g., in the substantia gelatinosa) and in the peripheral sensoryneurons. Kappa opioid receptors play a role in analgesia, anticonvulsanteffects, depression, dissociative/hallucinogenic effects, diuresis,dysphoria, miosis, neuroprotection, sedation, and stress.

The delta (δ) opioid receptor (DOR) is found in the brain (e.g., in thepontine nuclei, amygdala, olfactory bulbs, deep cortex) and in theperipheral sensory neurons. Delta opioid receptors play a role inanalgesia, antidepressant effects, convulsant effects, physicaldependence, and may modulate μ-opioid receptor-mediated respiratorydepression

The macrocyclic peptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp] (SEQ IDNO: 1) exhibits mixed opioid agonist/kappa opioid receptor (KOR)antagonist activity in vivo and is a promising lead compound fordeveloping novel therapeutics for the treatment of pain and drug abuse.We synthesized analogs of this lead peptide to explore the influence ofthe aromatic residues on the analogs' opioid activity profiles.

SUMMARY

Disclosed herein are cyclic tetrapeptide compounds, pharmaceuticalcompositions, and methods of using such pharmaceutical compositions. Thecyclic tetrapeptide compounds and pharmaceutical compositions disclosedherein are potent analgesics active in several pain models with, in somecases, decreased tolerance and reduced likelihood to induce addictionrelative to other known opiates.

In an embodiment, a cyclic tetrapeptide having a sequence ofcyclo[Phe-Pro-Phe-Trp] (SEQ ID NO: 1) having a structure of Formula 1 ora derivative thereof is disclosed.

In the illustrated structure of Formula 1, the wavy lines in the aminoacid sidechains indicate the existence of an unidentified stereocenter.Each of these amino acid stereocenters can be defined as ‘D’ or ‘L’using the standard notation for amino acids. Accordingly, the cyclictetrapeptide of Formula 1 may have an amino acid configuration of one ofLDDL, DDLL, DDDL, DDLD, LDDD, LLLL, DDDD, LLDL, DLLL, DLDL, LLLD, LLDD,DLDD, or DLLD. A cyclic tetrapeptide having an amino acid configurationof LDLL is specifically excluded from the claims herein. The naturallyoccurring cyclic tetrapeptide CJ-15,208 has the amino acid configurationof LDLL.

Preferably, the cyclic tetrapeptide of Formula 1 has an amino acidconfiguration of one of DDLL, DDDL, LDDD, DDLD, or DDDD. Morepreferably, the cyclic tetrapeptide of Formula 1 has an amino acidconfiguration of one of DDLL, DDDL, LDDD or DDDD. Most preferably, thecyclic tetrapeptide of Formula 1 has an amino acid configuration of DDDLor DDDD.

In another embodiment, a pharmaceutical composition is disclosed. Thepharmaceutical composition includes a pharmaceutically acceptableexcipient or carrier, and a therapeutically effective amount of thecyclic tetrapeptide of Formula I having an amino acid configuration ofone of LDDL, DDLL, DDDL, DDLD, LDDD, LLLL, DDDD, LLDL, DLLL, DLDL, LLLD,LLDD, DLDD, or DLLD. Preferably, the cyclic tetrapeptide of Formula 1has an amino acid configuration of one of DDLL, DDDL, LDDD, DDLD, orDDDD. More preferably, the cyclic tetrapeptide of Formula 1 has an aminoacid configuration of one of DDLL, DDDL, LDDD or DDDD. Most preferably,the cyclic tetrapeptide of Formula 1 has an amino acid configuration ofDDDL or DDDD.

In another embodiment, a method for treating pain is disclosed. Themethod includes administering to a subject a therapeutically effectiveamount of a pharmaceutical composition containing a cyclic tetrapeptideof Formula I having an amino acid configuration of one of LDDL, DDLL,DDDL, DDLD, LDDD, LLLL, DDDD, LLDL, DLLL, DLDL, LLLD, LLDD, DLDD, orDLLD. Preferably, the cyclic tetrapeptide of Formula 1 has an amino acidconfiguration of one of DDLL, DDDL, LDDD, DDLD, or DDDD. Morepreferably, the cyclic tetrapeptide of Formula 1 has an amino acidconfiguration of one of DDLL, DDDL, LDDD or DDDD. Most preferably, thecyclic tetrapeptide of Formula 1 has an amino acid configuration of DDDLor DDDD.

These and other objects and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only illustrated embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1 illustrates an analgesic effect of a cyclic tetrapeptide in aformalin assay;

FIG. 2 illustrates an analgesic effect of a cyclic tetrapeptide in anacetic acid stretching assay;

FIG. 3A-3B illustrate an analgesic effect of cyclic tetrapeptides in anerve constriction assay;

FIG. 4 illustrates that administration of the cyclic tetrapeptides doesnot induce drug seeking behavior;

FIG. 5 illustrates that administration of selected cyclic tetrapeptidesprevents stress-induced reinstatement of cocaine seeking behavior.

DETAILED DESCRIPTION

Disclosed herein are cyclic tetrapeptide compounds, pharmaceuticalcompositions, and methods of using such pharmaceutical compositions. Thecyclic tetrapeptide compounds and pharmaceutical compositions disclosedherein are potent analgesics active in several pain models withgenerally decreased tolerance and reduced likelihood to induce addictionrelative to other known opiates.

Recently, it has been shown that a cyclic tetrapeptide, CJ-15,208 andhaving Formula 1, can interact with opioid receptors (U.S. Pat. No.5,885,959). CJ-15,208 (i.e., cyclo[Phe-D-Pro-Phe-Trp]) (SEQ ID NO: 1)appears to be the natural product (see, Ross et al., TetrahedronLetters, 2010, 51, 5020-5023). The interaction was indicated to provideanalgesic activity, which can be attributed to being an antagonist.Accordingly, the true functionality of CJ-15,208 was not properlycharacterized. It has now been found that CJ-15,208 has weak antagonistactivities at kappa opioid receptors (KOR) in vivo. The structure ofCJ-15,208 is shown below.

It has now been found that cyclic tetrapeptides that are stereochemicalisomers of CJ-15,208 can have superior opioid receptor agonist activity(i.e., pain relieving activity), and thereby can be useful as analgesicsfor the treatment of inflammatory and neuropathic pain. It has furtherbeen found that that selected cyclic tetrapeptides disclosed herein showdecreased tolerance and reduced likelihood to induce addiction relativeto other known opiates.

The cyclic tetrapeptides discussed herein have a structure of Formula 1or a derivative thereof.

In the illustrated structure of Formula I, the wavy lines in the aminoacid sidechains indicate the existence of an undefined stereocenter.Each of these stereocenters can be defined as ‘D’ or ‘L’ according tostandard amino acid convention. Accordingly, the cyclic tetrapeptide ofFormula I may have an amino acid configuration of one of LDDL, DDLL,DDDL, DDLD, LDDD, LLLL, DDDD, LLDL, DLLL, DLDL, LLLD, LLDD, DLDD, orDLLD. Discussions of analogs or derivatives herein expressly excludeCJ-15,208, which has an amino acid configuration of LDLL.

As used herein, “derivatives” are considered to include R changes to theamino acid side chains of amino acids of the cyclic tetrapeptide ofFormula 1. Derivatives are considered to include atom or substituentexchanges such as one or more hydrogen atoms being substituted with analkyl, halogen, hydroxy, amine, combinations thereof, or the like. Forinstance, a number of phenylalanine (Phe) and tryptophan (Trp)derivatives are known in the art. Accordingly, Phe and Trp derivativesmay include, but are not limited to, changes where one or more hydrogen,carbon or hetero atoms of the side chain are replaced with one or moreof halo, amino, straight-chain alkyl, branched alkyl, alkoxy, nitro,halo alkyl, cyano, or hydroxy.

The cyclic tetrapeptides disclosed and claimed herein are completelydifferent in structure from known opioid ligands, which are typicallyeither peptide or nonpeptide. For instance, the cyclic tetrapeptideslack a free N-terminal basic amine found in endogenous opioid peptideligands and other peptides with high affinity for opioid receptors.Interestingly, changing the stereochemistry of the Trp residue, which isbelieved to be important for KOR binding, results in a new analog whichretains KOR affinity comparable to the parent CJ-15,208, which issurprising and unexpected. In both CJ-15,208 and the D-Trp analog ofCJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp] (SEQ ID NO: 1) (Formula 2802)). InU.S. Pat. No. 8,809,278 it was shown that the substitution of Phe by Alaenhances affinity of the cyclic tetrapeptide for KOR by about 4- to10-fold. In contrast to linear peptides, the cyclic tetrapeptides aremetabolically stable to proteases and exhibit enhanced membranepenetration (e.g. through the blood-brain barrier).

The cyclic tetrapeptides shown below in Tables 1 and 2 can be used asanalgesics for the treatment of pain. In addition, these cyclictetrapeptides, which are opioid receptor ligands, can be used in theprevention, inhibition, and/or treatment of drug abuse, specifically totreat, inhibit, and/or prevent stress-induced drug seeking behavior.Cyclic tetrapeptides can be metabolically stable, and can be activefollowing systemic (e.g., oral (per os), subcutaneous, s.c.,intravenous, i.v., or the like) administration. The cyclic tetrapeptideantagonists can block stress-induced reinstatement of drug-seekingbehavior following systemic administration.

In one embodiment, the cyclic tetrapeptides disclosed and claimed hereincan be used as analgesics. Opioid receptor agonists are drug compoundsthat activate opioid receptors. Full agonist opioids activate the opioidreceptors fully resulting in the full opioid effect. Examples of fullagonists are heroin, oxycodone, methadone, hydrocodone, morphine, opiumand others. The tetrapeptides disclosed and claimed are demonstratedherein to have analgesic properties similar to traditional opioids. Assuch, the cyclic tetrapeptides disclosed and claimed herein can be usedto treat and/or inhibit pain treatable by an analgesic.

The functionality of cyclic tetrapeptides was studied by preparingstereochemical isomers of CJ-15,208, and testing the interactions withopioid receptors in vitro and in various pain models in vivo. It has nowbeen found that stereochemical changes to the amino acids in the cyclictetrapeptide actually increased analgesic effects in relevant painassays in a surprising and unexpected manner.

Because opioid receptor ligands are known to have central nervous systemto effects, the cyclic tetrapeptides disclosed and claimed herein canalso be used for the treatment of drug abuse by blocking stress-inducedreinstatement of drug-seeking behavior following systemic administration(see, e.g., FIG. 5). Drugs included in this are at least cocaine,alcohol, methamphetamines, amphetamines, opioids (e.g., narcotic opioidalkaloids, morphine, codeine, heroin, oxycodone, hydrocodone, and anybenzylisoquinoline alkaloid; synthetic opiates, fentanyl, meperidine andmethadone), and the like. Accordingly, the cyclic tetrapeptides could beused as a prophylactic to prevent relapse to drug addiction. It isexpected that the cyclic tetrapeptides may also be active in models fortreatment of drugs of abuse other than cocaine, specifically opiates andamphetamine, and possibly others (e.g., alcohol and nicotine).

Likewise, in one embodiment, the cyclic tetrapeptides disclosed hereinmay be able to be used to treat, inhibit, and/or prevent depression.Based on the activity of small molecule opioid receptor ligands, thecyclic tetrapeptides are expected to exhibit antidepressant activity.Similarly, the cyclic tetrapeptides can be used for treating,inhibiting, and/or preventing anxiety.

Structures of stereochemical isomers of CJ-15,208

JVA # Compound Structure 2801 (LDLL)* cyclo[Phe-D- Pro-Phe-Trp] (SEQ IDNO: 1)

2802 (LDLD) cyclo[Phe-D- Pro-Phe-D-Trp] (SEQ ID NO: 1)

3606 (LDDL) cyclo[Phe-D- Pro-D-Phe-Trp] (SEQ ID NO: 1)

3605 (DDLL) cyclo[D-Phe-D- Pro-Phe-Trp] (SEQ ID NO: 1)

3607 (DDDL) cyclo-[D-Phe-D- Pro-D-Phe-Trp] (SEQ ID NO: 1)

3609 (DDLD) cyclo[D-Phe-D- Pro-Phe-D-Trp] (SEQ ID NO: 1)

3608 (LDDD) cyclo[Phe-D- Pro-D-Phe- D-Trp] (SEQ ID NO: 1)

3612 (LLLL, all L) cyclo[Phe-Pro- Phe-Trp] (SEQ ID NO: 1)

3613 (DDDD, all D) cyclo[D-Phe-D- Pro-D-Phe- D-Trp] (SEQ ID NO: 1)

3614 (DLDD) cyclo[D-Phe- Pro-D-Phe- D-Trp] (SEQ ID NO: 1)

3636 (LLDL) cyclo[Phe-Pro- D-Phe-Trp] (SEQ ID NO: 1)

3637 (DLLL) cyclo[D-Phe- Pro-Phe-Trp] (SEQ ID NO: 1)

3638 (DLDL) cyclo[D-Phe- Pro-D-Phe-Trp] (SEQ ID NO: 1)

3639 (LLLD) cyclo[Phe-Pro- Phe-D-Trp] (SEQ ID NO: 1)

3640 (LLDD) cyclo[Phe-Pro- D-Phe-D-Trp] (SEQ ID NO: 1)

3641 (DLLD) cyclo[D-Phe- Pro-Phe-D-Trp] (SEQ ID NO: 1)

*The shorthand notation in parentheses indicates the amino acidconfigurations within the peptide

The cyclic peptide stereochemical isomers of CJ-15,208 disclosed hereinand shown in Table 1 were synthesized by a combination of solid phasesynthesis with cyclization in solution.

The peptides of Table 1 shown below in Tables 3 and 4 were evaluated inradioligand binding assays for mu, kappa and delta opioid receptoraffinities in vitro, and in the mouse 55° C. warm water tail withdrawalassay in vivo for antinociception and opioid antagonist activity.Additionally, peptides 3605, 3607, and 3608 showed significant analgesicproperties when evaluated in other pain models (FIGS. 1-3B), andpeptides 3605 and 3607 showed no evidence of inducing drug seekingbehavior when evaluated in a conditioned place response assay (FIG. 4).

TABLE 3 In vitro receptor binding affinities^(a) K_(i) (nM ± SEM)^(b)Selectivity JVA # KOR MOR DOR^(c) KOR/MOR/DOR 2801 35.4 ± 3.6 (4) 619 ±87 (3) 1720 ± 350 (5) 1/18/49 (CJ-15,208) 16.9 ± 5.2 (3) 200 ± 54 (2)1/12/102 3605 5120 ± 690 >10,000 (4) >10,000 3606 362 ± 51 3920 ± 200(3) >10,000 1/11/>27 3607 2560 ± 480 7780 ± 410 >10,000 1/3/>3.8 280230.6 ± 3.4 (5) 259 ± 29 4190 ± 860 (6) 1/8.5/137 ([D-Trp]  7.0 ± 4.8 (3)1/37/599 CJ-15,208) 3609 >10,000 >10,000 >10,000 — 3608 353 ± 19  5800 ±1450 >10,000 1/16/>28 3613 2010 ± 270 >10,000 >10,000 1/>4.9/.4.9^(a)None of these compounds exhibit agonist activity at KOR or MOR inthe GTPγS assay at 10 μM.

The cyclic tetrapeptide stereoisomers of CJ-15,208 have relatively lowaffinity for KOR, MOR, and DOR. Only two stereoisomers, 3606 and 3608,exhibit K_(i)<1 μM and these stereoisomers show very low affinity forMOR (K_(i)>1 μM) and negligible affinity for DOR. None of thesestereoisomers exhibit evidence of agonist activity in the in vitrofunctional assay.

Nonetheless, the analogs generally exhibited potent antinociception invivo comparable to CJ-15,208 following both intracerebroventricular(i.c.v.) and oral (per os) administration in spite of reductions inopioid receptor affinities in vitro (Table 4).

Antinociceptive and opioid antagonist activity:

TABLE 4 ED₅₀ (95% CI) i.c.v. p.o. Receptors Antagonism JVA # (nmol)(mg/kg) involved (dose, nmol) 2801 1.74 3.49 KOR, MOR KOR (CJ-15,208)(0.62-4.82) (1.98-5.73) 3605 0.75 7.62 KOR, MOR, KOR (30), (0.36-1.44)(5.12-12.2) DOR DOR (100) 3606 Partial agonist >30 (~45% KOR, MOR None(100) (~60% max) response @ 30 mg/kg) 3607 1.00 4.12 KOR, DOR None (30)(0.64-1.60) (3.30-5.31) (not MOR) 2802 ~40% @ ~25% @ KOR KOR ([D-Trp] 30nmol 60 mg/kg CJ-15,208) 3609 2.39 ≥10 mg/kg DOR, KOR None (30)(1.40-4.56) (~60% at (not MOR) 30 mg/kg) 3608 0.56 4.72 KOR, MOR, DOR(100) (0.38-0.91) (3.70-6.39) DOR 3613 0.51 2.47 DOR, MOR DOR (30)(0.39-0.67) (0.97-4.71) (not KOR)

All of these stereoisomers of 2801 and 2802 except 3606 are potent fullagonists in the antinociception assay after central administration. Allof these stereoisomers except 3606 and 3609 are potent full agonistsafter oral administration. Interestingly, these in vivo results do notcorrelate with the in vitro results. This indicates that themechanism(s) of opioid receptor agonism in vivo may be complex.

The evidence showed that multiple opioid receptors contributed to theantinociception of each analog. These include compounds where mu opioidreceptors (MOR) are not involved (3607 and 3609), and compounds werekappa opioid receptors (KOR) are not involved (3613). In contrast to theparent peptides (i.e., 2801 and 2802), only one of the analogs exhibitedKOR antagonism in vivo. Only one stereoisomer, 3605, exhibits KORantagonist activity, but the dose-response of the antagonism of thiscompound is unusual. Three of the stereoisomers, 3605, 3608, and 3613,exhibit DOR antagonist activity. In interesting comparison to the othercyclic tetrapeptides, compound 3613 (DDDD) is a mu opioid receptor (MOR)agonist and delta opioid receptor (DOR) antagonist. MOR agonists/DORantagonists are of great interest because of their decreased liabilityprofile (decreased addiction potential and liability profile such astolerance) compared to MOR agonists like morphine. In addition, compound3613 is effective in preventing stress-induced reinstatement of cocaineseeking behavior in the conditioned place preference assay (FIG. 5).This is a surprising result given that compound 3613 does not appear toto have KOR antagonist activity.

Referring now to FIGS. 1-3B, data from various assays show thatcompounds 3605, 3607, and 3708 show activity against pain in a number ofmodels. These models are based on the formalin assay, the acetic acidstretching assay, and the nerve constriction assay. The formalin andacetic acid stretching assays are indicative of activity againstinflammatory pain (i.e., nociceptive pain) and the nerve constrictionassay is indicative of activity against neuropathic pain.

Inflammatory pain (i.e., nociceptive pain) is caused by stimulation ofperipheral nerve fibers that respond only to stimuli approaching orexceeding harmful intensity (nociceptors), and may be classifiedaccording to the mode of noxious stimulation. The most common categoriesbeing “thermal” (e.g., heat or cold), “mechanical” (e.g., crushing,tearing, shearing, etc.) and “chemical” (e.g., salt or iodine in a cut).

Neuropathic pain is caused by damage or disease affecting any part ofthe nervous system involved in bodily feelings (the somatosensorysystem). Peripheral neuropathic pain is often described as “burning”,“tingling”, “electrical”, “stabbing”, or “pins and needles”.

FIG. 1 demonstrates that a 10 mg/kg oral administration of 3607 iseffective in alleviating pain in the formalin assay as compared tointraperitoneal administration of saline. It entails the injection of adilute solution of formalin into the surface of the rodent's hindpaw,followed by the scoring of stereotypical behaviors such as flinching,licking, and biting of the affected hindpaw. The behaviors last forapproximately 1 hour, with the early or acute stage (directly afterinjection) reflecting direct activation of nociceptors and the late ortonic phase (15 to 20 minutes after the injection) reflectinginflammation. Administration of 3607 produced a significant reduction inthe time spent licking after formalin administration.

FIG. 2 demonstrates that administration of 3607 was effective for painreduction in the acetic acid stretching assay. The acetic acidstretching assay is a model for visceral pain and is the most popularchemical assay of nociception. In the assay, subjects are given asubcutaneous (s.c.) injection of drug or vehicle 60 min before anintraperitoneal (i.p.) injection of 0.6% acetic acid. Afteradministration of the acetic acid, the subjects are placed in clearcages (11×7×5 in) and scored for abdominal stretches during a 20 minobservation period. Stretching was defined as body contortions, bellypressing, and extension of the hind limbs from which visceralnociception was inferred. Administration of 10 mg/kg and 30 mg/kg of3607 produced significant antinociception as compared to administrationof morphine. And while the effect of 3607 was not as pronounced as withmorphine, the observed reductions in stretching were significant,indication that 3607 has a significant pain reduction effect.

Referring now to FIGS. 3A-3B, FIGS. 3A-3B demonstrate that 3607 (FIG.3A) and 3605 and 3608 (FIG. 3B) are additionally active in the nerveconstriction assay for neuropathic pain. In the nerve constrictionassay, a nerve of a subject rodent is chronically constricted or cuffedand then the animal is assessed for response to pain stimuli such a pawwithdrawal. FIG. 3A demonstrates that 3607 at 3 mg/kg, 10 mg/kg, and 30mg/kg produces significant relief of neuropathic pain, as compared to 50mg/kg gabapentin. Gabapentin is an accepted treatment for neuropathicpain. FIG. 3B demonstrates that 3605 and 3608 at 10 mg/kg p.o. producesignificant relief of neuropathic pain, as compared to 50 mg/kg i.p.gabapentin and compound 2801.

Interestingly, when screened in an acute tolerance model, theantinociceptive tolerance observed varied markedly among the peptides,from no significant display of tolerance to a rightward shift in thedose-response curve equal to or greater than that exhibited by morphine.

Changes in the macrocyclic tetrapeptide structure were well toleratedwith retention of antinociceptive activity, while the KOR antagonism wasvery sensitive to changes in the aromatic residues. The identificationof compounds with potent antinociception that exhibit decreasedtolerance in the initial screening is a promising development in thesearch for potential analgesics with improved liability profiles.

Liability Testing:

TABLE 5 Acute Locomotion Respiratory Tolerance Rotorod (CLAMS,depression ED₅₀ (10 mg/kg, 10 mg/kg, 10 mg/kg JVA # shift^(a) p.o.)p.o.) p.o.) 2801 1.73 ✓ (10 and 60 (CJ-15,208) mg/kg) 3605 19.5-21.5 ✓(sig. ✓ Sig. resp. sedation depression @ 10 mg/kg, but not 30, mg/kg)3606 Substantial ✓ Sig. hypo- ✓ shift^(c) locomotion 3607 5.19 ✓ ✓ ✓2802 ✓ ([D-Trp] (60 mg/kg) CJ-15,208) 3609 1.87 ✓ (sig Sig. hypo- Resp.sedation locomotion depression at 40-60 min only) 3608 9.87 ✓ Sig. hypo-Resp. locomotion depression 3613 2.55 ✓ ✓ ✓ ^(a)Morphine sulfate causes9.2 fold shift in the ED₅₀ under these conditions; ^(b)✓ indicatestested (if no other comment then no significant effect); ^(c)Cannotreliably calculate ED₅₀ values because compound is a partial agonist.

As shown in Table 5, the tested compounds performed generally well inliability testing assays. In acute tolerance assays, the shift in theED₅₀ values varied markedly from minimal (3609 and 3613) to more thanmorphine (3605). This was surprising. In a rotorod test for sedation andmotor coordination, only in the case of 3605 were effects similar to theKOR selective agonist U50,488, suggesting sedation.

None of the compounds showed hyperlocomotion like morphine and other MORagonists. Given that most of the analogs did not show evidence ofsedation in the rotorod assay, the decreases in locomotion observed areprobably not due to sedation. Some analogs, namely 3605, 3608, and 3609,did show evidence of respiratory depression at 10 mg/kg, p.o.

Based on the liability profiles the most promising stereoisomers appearto be 3607 and 3613.

Referring now to FIG. 4, it was observed that compounds 3605 and 3607 donot show evidence of conditioned place preference (drug seekingbehavior) or conditioned place aversion following oral administration.Compounds 3605 and 3607 were compared to morphine and U50,488, whichshow significant conditioned place preference (drug seeking behavior)and conditioned place aversion, respectively.

L-Proline containing peptides:

In vitro results: These stereoisomers show low affinity for KOR (K_(i)>1μM). They also generally show low affinity for MOR, with only twostereoisomers exhibiting a K_(i)<1 μM. These compounds exhibitnegligible affinity for DOR

In vivo results:

To date most of these stereoisomers have only been evaluated followingi.c.v. administration. Only selected analogs have been evaluated atdoses >10 nmol.

Stereoisomers with demonstrated antinociceptive activity:

3612: ED₅₀=3.52 (2.01-6.42) nmol i.c.v., 4.28 (3.56-5.27) mg/kg p.o.(KOR, MOR and DOR involved)

3638: ED₅₀=8.93 (1.96-19.5) nmol i.c.v.

3639: ED₅₀=49.7 (34.8-69.8) nmol i.c.v.

No or only weak antagonist activity has been detected for thesestereoisomers to date, but some analogs have only been tested at a lowerdose (10 nmol i.c.v.)

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope. All references recitedherein are incorporated herein by specific reference in their entirety.

What is claimed is:
 1. A cyclic tetrapeptide comprising a structure ofFormula 1 with an amino acid sequence of cyclo[Phe-Pro-Phe-Trp] (SEQ IDNO: 1) or a derivative thereof, wherein the cyclic tetrapeptidecomprises an amino acid configuration selected from the group consistingof: [LDDL], [DDLL], [DDDL], [DDLD], [LLLL], [LLDL], [DLLL], [DLDL],[LLLD], [LLDD], [DLDD], and [DLLD], and wherein amino acid configurationof the cyclic tetrapeptide does not comprise [LDLL]


2. The cyclic tetrapeptide of claim 1, wherein the amino acidconfiguration of the cyclic tetrapeptide is selected from the groupconsisting of; [DDLL], [DDDL], and [DDLD].
 3. The cyclic tetrapeptide ofclaim 1, wherein the amino acid configuration of the cyclic tetrapeptideis selected from the group consisting of: [DDLL] and [DDDL].
 4. Apharmaceutical composition, comprising: a pharmaceutically acceptableexcipient or carrier; and a therapeutically effective amount of thecyclic tetrapeptide of claim
 1. 5. The pharmaceutical composition ofclaim 4, wherein the amino acid configuration of the cyclic tetrapeptideis selected from the group consisting of: [DDLL], [DDDL], and [DDLD]. 6.The pharmaceutical composition of claim 4, wherein the amino acidconfiguration of the cyclic tetrapeptide is selected from the groupconsisting of: [DDLL] and [DDDL].
 7. The pharmaceutical composition ofclaim 4, wherein the amino acid configuration of the cyclic tetrapeptidecomprises [DDDL].
 8. The pharmaceutical composition of claim 4, whereinthe therapeutically effective amount of the cyclic tetrapeptide iseffective for analgesia.
 9. The pharmaceutical composition of claim 4,wherein the therapeutically effective amount of the cyclic tetrapeptideis effective for treating inflammatory pain.
 10. The pharmaceuticalcomposition of claim 4, wherein the therapeutically effective amount ofthe cyclic tetrapeptide is effective for treating inflammatory pain andneuropathic pain.
 11. The pharmaceutical composition of claim 5, whereinthe therapeutically effective amount of the cyclic tetrapeptide iseffective for one or more of analgesia, treating inflammatory pain,treating neuropathic pain, or combinations thereof.
 12. Thepharmaceutical composition of claim 6, wherein the therapeuticallyeffective amount of the cyclic tetrapeptide is effective for one or moreof analgesia, treating inflammatory pain, treating neuropathic pain, orcombinations thereof.
 13. The pharmaceutical composition of claim 7,wherein the therapeutically effective amount of the cyclic tetrapeptideis effective for one or more of analgesia, treating inflammatory pain,treating neuropathic pain, or combinations thereof.
 14. A method fortreating and/or preventing pain, comprising administering to a subject atherapeutically effective amount of a pharmaceutical composition ofclaim
 4. 15. The method of claim 14, wherein the amino acidconfiguration of the cyclic tetrapeptide is selected from the groupconsisting of: [DDLL], [DDDL], and [DDLD].
 16. The method of claim 14,wherein the amino acid configuration of the cyclic tetrapeptide isselected from the group consisting of: [DDLL] and [DDDL].
 17. The methodof claim 14, wherein the amino acid configuration is of the cyclictetrapeptide comprises [DDDL].